Chen Jing, Huang Chunlan, Wang Jingjing, Zhou Hui, Lu Yingying, Lou Lihong, Zheng Junyuan, Tian Ling, Wang Xingpeng, Cao Zhongwei, Zeng Yue
Department of Gastroenterology, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Shanghai Key Laboratory of Pancreatic Diseases, Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
PLoS One. 2017 Apr 25;12(4):e0176583. doi: 10.1371/journal.pone.0176583. eCollection 2017.
Intestinal barrier dysfunction plays an important role in acute necrotizing pancreatitis (ANP) and intestinal microbiota dysbiosis was involved in intestinal barrier failure. Paneth cells protect intestinal barrier and are associated with intestinal microbiota. Here, we investigated changes in intestinal microbiota and antimicrobial peptides of Paneth cells in ileum during ANP.
Rats with ANP were established by retrograde injection of 3.5% sodium taurocholate into biliopancreatic duct and sacrificed at 24h and 48h, respectively. Injuries of pancreas and distal ileum were evaluated by histopathological score. Intestinal barrier function was assessed by plasma diamine oxidase activity (DAO) and D-lactate. Systemic and intestinal inflammation was evaluated by TNFα, IL-1β and IL-17A concentration by ELISA, respectively. 16S rRNA high throughput sequencing on fecal samples was used to investigate the changes in intestinal microbiota in the ANP group at 48h. Lysozyme and α-defensin5 were measured by real-time PCR, western blot and immunofluoresence.
ANP rats had more severe histopathological injuries in pancreas and distal ileum, injured intestinal barrier and increased expression of TNFα, IL-1β and IL-17A in plasma and distal ileum compared with those of the sham-operated (SO) group. Principal component analysis (PCA) showed structural segregation between the SO and ANP groups. Operational taxonomic unit (OTU) number and ACE index revealed decreased microbiota diversity in the ANP group. Taxonomic analysis showed dysbiosis of intestinal microbiota structure. At phyla level, Saccharibacteria and Tenericutes decreased significantly. At genus level, Escherichia-Shigella and Phascolarctobacterium increased significantly, while Candidatus_Saccharimonas, Prevotellaceae_UCG-001, Lachnospiraceae_UCG-001, Ruminiclostridium_5 and Ruminococcaceae_UCG-008 decreased significantly. Lysozyme and α-defensin5 mRNA expression levels decreased significantly in ANP group at 48h. Protein expression of lysozyme decreased in ANP groups at 24h and 48h. Meanwhile, the relative abundance of Escherichia-Shigella correlated inversely with the decrease in lysozyme.
The disorder in intestinal microbiota and decreases of Paneth cell antimicrobial peptides might participate in the pathogenesis of intestinal barrier dysfunction during ANP.
肠道屏障功能障碍在急性坏死性胰腺炎（ANP）中起重要作用，肠道微生物群失调与肠道屏障功能衰竭有关。潘氏细胞可保护肠道屏障并与肠道微生物群相关。在此，我们研究了ANP期间回肠中肠道微生物群和潘氏细胞抗菌肽的变化。
通过向胆胰管逆行注射3.5%牛磺胆酸钠建立ANP大鼠模型，并分别在24小时和48小时处死。通过组织病理学评分评估胰腺和回肠远端的损伤情况。通过血浆二胺氧化酶活性（DAO）和D-乳酸评估肠道屏障功能。分别通过ELISA法检测TNFα、IL-1β和IL-17A浓度来评估全身和肠道炎症。对粪便样本进行16S rRNA高通量测序，以研究ANP组在48小时时肠道微生物群的变化。通过实时PCR、蛋白质印迹和免疫荧光法检测溶菌酶和α-防御素5。
与假手术（SO）组相比，ANP大鼠的胰腺和回肠远端组织病理学损伤更严重，肠道屏障受损，血浆和回肠远端中TNFα、IL-1β和IL-17A的表达增加。主成分分析（PCA）显示SO组和ANP组之间存在结构分离。操作分类单元（OTU）数量和ACE指数显示ANP组中微生物群多样性降低。分类学分析显示肠道微生物群结构失调。在门水平上，糖菌门和柔膜菌门显著减少。在属水平上，埃希氏菌-志贺氏菌属和袋形杆菌属显著增加，而候选糖单胞菌属、普雷沃氏菌科_UCG-001、毛螺菌科_UCG-001、瘤胃梭菌属_5和瘤胃球菌科_UCG-008显著减少。ANP组在48小时时溶菌酶和α-防御素5 mRNA表达水平显著降低。ANP组在24小时和48小时时溶菌酶蛋白表达降低。同时，埃希氏菌-志贺氏菌属的相对丰度与溶菌酶的降低呈负相关。
肠道微生物群紊乱和潘氏细胞抗菌肽减少可能参与ANP期间肠道屏障功能障碍的发病机制。
